Apparatus and method for growing anaerobic microorganisms

ABSTRACT

An apparatus for growing anaerobic microorganisms is provided having a dish top that contains a sealing ring upon which the media surface in the dish bottom rests when the apparatus is inverted. The contact between the sealing ring and the media surface forms a seal that traps the gas in the headspace between the media surface and the inside of the dish top. A oxygen reducing agent can also be incorporated into the media together, in some instances, with a substrate which react with oxygen in the media and with oxygen in the headspace thereby creating an environment suitable for growing anaerobic, microaerophilic and facultative anaerobic microorganisms.

BACKGROUND OF THE INVENTION

The present invention relates to an apparatus and method for growinganaerobic microorganisms. The apparatus is comprised of a speciallydesigned culture dish which can be reconfigured such as by inverting thedish to produce an anaerobic environment. An oxygen reducing agent suchas a biocatalytic oxygen reducing agent can also be incorporated intothe media present in the apparatus together, in some circumstances, witha substrate. The biocatalytic oxygen reducing agent and the substratepresent in the media react with oxygen enclosed in the culture dish tocreate an environment suitable for growing and maintaining anaerobicmicroorganisms.

Microorganisms are important to our well being. This is evident inhealthcare, agriculture and industry. To be able to simply and quicklyisolate and grow microbes is economically important. For example, beingable to quickly and specifically isolate and identify a microberesponsible for infection is important in human health care field. Thisbasic technique is also important in the agriculture industry. Largescale processing of food requires constant microbial monitoring. Thespeed and efficiency at which this can be done determines the length oftime finished food products must be held in storage before they can bedistributed for sale.

Control of the environment is necessary for control of microbial growth.In particular, control of oxygen content in the immediate environment iscrucial for microbial growth. Microorganisms can be divided into groupsbased on their need for, and tolerance of, oxygen. There are those thatrequire oxygen to grow. These are "aerobes". Some microorganisms areable to grow with or without oxygen. These are "facultative anaerobes".Another group of microorganisms can grow only in the presence of verylow levels of oxygen. These are the "microaerophiles". Finally, somemicroorganisms can not tolerate oxygen. They are inhibited by it or maybe killed by it. These are the "anaerobes".

This fundamental property of microorganisms, that is their ability togrow in or tolerate oxygen, is used daily to isolate, grow, andmanipulate them. One basic technique in microbiology, is the platingmethod. This generally involves the use of a dish, developed by Petri(i.e. "Petri dish") in 1880's, and solidified (agar or gelatin-based)medium.

A Petri dish is usually a round, shallow, flat-bottomed, glass orplastic dish (often e.g. 10 cm diameter) with a vertical side, thatcooperates with a similar, slightly larger structure which forms aloosely-fitting lid. Petri dishes are used in microbiology, e.g., forthe preparation of plates.

The purpose of the Petri dish is to provide a controlled environment forselectively growing microbes. The dish is sterilized and designed tomaintain a sterile environment inside while freely exchanging gases,normally air, with the outside environment.

The medium utilized in conjunction with the Petri dish can be formulatedto provide a necessary and selective environment for a specificmicroorganism. Solid medium in a Petri dish can be prepared usingaseptic technique by pouring sterile molten or liquid (agar- orgelatin-based) medium into a Petri dish to a depth of 3-5 mm andallowing it to set. Generally, freshly poured plates to be used forseparation and/or generation of microbes should be left for 30 minutesin a 45° C. hot-air incubator with the lid partly off so that thesurface moisture can evaporate. Such "drying" before inoculationprevents unwanted spreading of the inoculum in the surface film of themoisture.

The solid medium surface inside the dish provides a place to growmicroorganisms. By inoculating (or "plating") the surface of the agar ina controlled way (i.e. "streaking"), single colonies of a microorganismcan be obtained. With this technique the microbiologist can separatemicrobes one from another. Isolation and purification is mandatory tofurther characterization and study. Using this dish design, amicrobiologist can isolate and grow the great majority of microorganismsknown today.

Working with microbes that are microaerophiles or anaerobes poses aproblem. The culture dishes for these microbes must be incubated in acontrolled gaseous environment that lacks oxygen, or at least most ofthe oxygen, found in air. This is done by placing the culture or Petridish containing medium inside a container that is sealed from theoutside atmosphere. For one or a few dishes, a sealable bag or jar(i.e., "Brewer Jar") is used (Becton Dickinson Microbiology Systems,1994 Catalog, p 89-p 94). In this case, chemicals and a catalyst (seeU.S. Pat. No. 4,287,306 issued Sep. 1, 1982 to Brewer entitled"Apparatus for Generation of Anaerobic Atmosphere") are placed insidethe container that, when activated chemically, reacts with the oxygen inthe container, thus removing it. The catalyst is necessary to bringabout the reaction at low temperatures in a short time.

In addition, for many culture dishes, a sealed table-top chamber can beused (Anaerobe Systems, San Jose, Calif.). This chamber is evacuated andflushed with inert gases, such as nitrogen and/or carbon dioxide.Sometimes chemicals and a catalyst are used to consume the oxygen insidethe chamber and fresh, inert gas is supplied as needed. Themicrobiologist works with the culture dishes inside of this chamberthrough ports fitted with gloves. A means is provided for introducingmaterials into and taking items out of the chamber without breaching theanaerobic environment inside.

Work with microaerophiles and anaerobes under these conditions is laborintensive, difficult, expensive, and time consuming. The microbiologistis often frustrated by having to wait for the slowest growing microbe inorder to retrieve all culture dishes from a bag or jar since once thebag or jar is opened, the microbes are exposed to oxygen. A failure inthe system can be catastrophic for all of the microbial isolates inside.

To overcome many of these problems (see U.S. Pat. No. 2,348,448 issuedMay 9, 1944 to Brewer entitled "Apparatus for the Cultivation ofAnaerobic and Microaerophilic Organisms") Brewer developed a culturedish lid (i.e., "Brewer Lid") that formed a seal between a ring insidethe lid with the agar or gelatin-based surface. Within the dish, a verysmall, defined headspace is formed by the lid and the agar surface. Ananaerobic environment is created inside this trapped headspace byreacting oxygen with chemical reducing agents, such as thioglycollate,incorporated in the medium. The limited volume of the headspace isimportant to the function of the Brewer Lid.

However, a number of drawbacks exist in the use of the Brewer Lid. Thecapacity and the rate for oxygen removal is limited by the sensitivityof the microorganism to the chemical reducing agent in the medium (see"Mechanism of Growth Inhibitory Effect of cysteine on Escherichia coli." of Kari, et al., J. Gen. Microbiol., 68, 1971, p. 349 and "Methods forGeneral and Molecular Bacteriology", Editor: Gerherdt, American Societyfor Microbiology, 1994, p. 146.). Moreover, the lid is made of heavyglass and is expensive. It is available today (Kimble Glass Company,Vineland, N.J.), but is not widely used because of serious limitationsthat include cost, handling difficulties, and poor response of anaerobicmicroorganisms.

Another limitation is caused by the material of construction. The glassBrewer Lid is made very heavy to insure a good seal between the ringinside the Brewer Lid and the agar surface. Cultures dish bottoms fittedwith the heavy Brewer Lid are not easy to handle or to move about. Theycan not be stacked inside an incubator. Thus, precious incubator spaceis wasted. Stacked dishes crush the agar medium of the lowest dishes inthe stack, because of the weight of the dishes above them. This causesthe headspace above the agar to collapse resulting in contact betweenthe inside of the Brewer Lid and the agar surface. When this happens,the microbial growth on the surface is spread out and separation ofindividual colonies is lost. Motile microbes will migrate and furtherfrustrate separation.

Because of their weight and material of construction, Brewer Lids do notlend themselves to commercial production of pre-made agar orgelatin-based plates. The commercial process requires assembly linefilling of the dishes, packaging the filled dishes in stacks, andhandling and storing these dishes. Pre-made agar plates are widely usedin clinical microbiological laboratories. This limitation of the BrewerLid is economically significant.

The headspace inside the Brewer Lid formed by the lid and agar surfaceis very small. This limited headspace is determined by the ability ofthe chemical reducing agent (H₂ S, cysteine, thioglycollate, etc.) toreduce oxygen in the headspace. The amount of chemical reducing agentused in the medium in turn is constrained by anaerobic microorganism'ssensitivity to it. The sum of these limitations is a very small headspace that imparts severe problems to the function of the Brewer Lid forits intended purpose, i.e. to grow anaerobic and microaerophilicmicroorganisms.

Another limitation of the Brewer Lid is that the very limited head spacecan not hold much moisture. Fresh agar medium is generally greater than98 percent water. Upon incubation, water in the medium evaporates andcondenses upon the upper surface of the inside of the lid. Thiscondensate can become sufficient to fall to the agar surface and toflood it. Under such conditions, the plate is ruined and can not be usedfor isolation and purification of the microbe.

The very limited headspace imposes still more limitations on the BrewerLid. No provision is made to incorporate CO₂ into the headspace abovethe agar surface. This is important for the rapid growth of somemicroorganisms and may be required by others. Yet this feature should bemade optional for the microbiologist, because for some uses of theculture dish the microbiologist may not want to include CO₂ in theheadspace. Reports show that CO₂ can change the pH of the medium itcontacts. This in turn can interfere with the determination ofsusceptibility to some antibiotics (see "Effect of CO₂ onSusceptibilities of Anaerobes to Erythromycin, Azithromycin,Clarithromycin, and Roxithromycin", Spangler, et al., Antimicrob. AgentsChemotherapy, 38, p. 20, 1994). Since CO₂ is generated in anaerobic jarsand bags by commercial catalysts products, this problem is commonlyencountered. CO₂ is a component of the gas used to flush anaerobicchambers and incubators too.

Another desired feature for a self contained culture dish is anindicator to show that the headspace is anaerobic. These features aredifficult to impossible to include in the Brewer Lid because of the verysmall space between inside the lid top and the agar surface.

Several attempts have been made to design a culture dish that provides aself-contained environment for growing anaerobic microorganisms (seeU.S. Pat. No. 2,701,229 issued Feb. 1, 1955 to Scherr entitled"Apparatus for the Cultivation of Microorganisms"; U.S. Pat. No.3,165,450 issued Jan. 12, 1965 to Scheidt entitled "Anaerobic CulturingDevice"; U.S. Pat. No. 4,294,924 issued Oct. 13, 1981 to Pepicelli, etal. entitled "Method and Container for Growth of AnaerobicMicroorganisms"; U.S. Pat. No. 4,299,921 issued Nov. 10, 1981 to Youssefentitled "Prolonged Incubation Microbiological Apparatus and FilterGaskets Thereof"; and U.S. Pat. No. 4,859,586 issued Aug. 8, 1989 toEisenberg entitled "Device for Cultivating Bacteria"). The fact that theBrewer Lid and none of these inventions are commonly or commerciallyavailable or used widely by microbiologists today, attest to theirlimitations and shortcomings. The need to simplify and reduce the costfor isolating and growing anaerobic and microaerophilic microorganismsstill exists today.

It is therefore an object of the present invention to provide animproved apparatus and method for cultivating and/or enumeratinganaerobic microorganisms which obviate the above-mentioned disadvantagesof the prior art.

Another object of the present invention is to provide an improvedanaerobic culturing apparatus which is extremely simple, inexpensive andeasy to use and wherein the proper anaerobic environment is produced andmaintained in an extremely efficient manner.

These and other additional objects and advantages of the presentinvention will become apparent from the following description of theinvention.

SUMMARY OF THE INVENTION

The present inventors have designed a novel culture apparatus or dish inorder to eliminate many of the difficulties observed in the prior art.It has been found that the use of the new culture dish (i.e., "OxyDish") together with an oxygen reducing agent (preferably a biocatalyticoxygen reducing agent) and, in some instances, a substrate, produces acontrolled, self-contained environment for isolating, enumerating,identifying and growing facultative aerobes, microaerophiles andanaerobes. The use of the specially designed culture dish along with anoxygen reducing agent makes possible the design and function of aculture dish that utilizes some features of the Brewer Lid, butovercomes its limitations and makes possible novel and improvedcharacteristics.

In this regard, the present invention is directed to a specificallydesigned culture dish with a dish top or cover that contains a sealingring on the inside upon which the solid media surface in the bottom dishrests when the dish is inverted to form a media-ring seal. The seal soformed traps the gas in the headspace between the media surface and theinside of the dish top or cover. In addition, an oxygen reducing agent,such as a biocatalytic oxygen reducing agent, can be incorporated intothe media present in the culture dish together, in some instances, witha substrate which reacts with oxygen in the media and the headspace tocreate an environment suitable for growing anaerobic microorganisms.

The preferred biocatalytic oxygen reducing agent (see "A Novel Approachto the Growth of Anaerobic Microorganisms" of Adler, et al., Biotechnol.Bioegn. Symp. 11, J. Wiley & Sons, New York, 1981, p. 533 and U.S. Pat.No. 4,476,224 issued Oct. 9, 1984 to Adler entitled "Material and Methodfor Promoting the Growth of Anaerobic Bacteria") utilized in theinvention is comprised of oxygen scavenging membrane fragments whichcontain an electron transport system which reduces oxygen to water inthe presence of a hydrogen donor. These oxygen scavenging membranefragments can be derived from the cytoplasmic membranes of bacteria(U.S. Pat. No. 4,476,224) and/or from the membranes of mitochondrialorganelles of a large number of higher non-bacterial organisms. Otherknown biocatalytic oxygen reducing agents such as glucose oxidase,alcohol oxidase, etc. can also be utilized.

The biocatalytic oxygen reducing agents suitable for use in theinvention are non-toxic to microorganisms. Being catalysts, they aredynamic and highly efficient at reducing the oxygen in the trappedheadspace in the specially designed culture dish. The biocatalyticoxygen reducing agents use substrates that are commonly found inmicrobiological media and that are natural to microorganisms to effectthis reaction. The products produced from this reaction are also naturaland non-toxic to microorganisms. The use of the biocatalytic oxygenreducing agents makes possible the opening and closing of this dishseveral times and the agents continue to reduce the oxygen trapped inthe headspace after each occurrence.

The culture dish ("OxyDish™") containing the oxygen reducing agentprovides a means to work with microorganisms free of the complicationsand expense of anaerobic bags, jars, incubators, or chambers. Each dishis light in weight and is designed to be stacked without crushing thesolid (agar or gelatin-based) medium in the lower dishes in the stack.The dishes can be made of low cost materials, preferably plastic, aredesigned to be readily molded, are sterilizable, and preferably can bedisposed after use. Because of the incorporation of a biocatalytic meansof removing oxygen, an enlarged headspace is possible. This enlargedheadspace relieves the moisture condensation problems encountered withthe Brewer Lid.

Moreover, the dish top of the culture dish in certain embodiments of thepresent invention, has a small dome or cavity designed to contain ananaerobic gas (such as CO₂) generating pad or indicator strips to showthe anaerobic state within the headspace of the closed culture dish. Avariation of this dish design provides for additional removal ofmoisture from the dish as needed by placing pores in the bottom of thedish base. This feature prevents the build-up of excess condensateinside the dish which leads to flooding of the agar media surface. Thepores are too small to let molten agar media flow out of the dish, yetthey provide an exit for moisture. Any oxygen intruding into the dishthrough these pores must pass through the media containing the oxygenreducing agent. This intruding oxygen is removed before it can diffuseto the top layer of media or into the headspace where it would interferewith growth of anaerobic microorganisms.

The culture dish, i.e., "OxyDish™" of the present invention, is designedfor automated preparation of agar or gelatin-based media platesnecessary for commercial production. When in the upright position, thedish can be readily filled with molten medium (such as a molten agar orgelatin-based media) without the sealing ring contacting the mediumsurface. When stored or used, the dish is placed into an invertedposition. In this position, a seal (i.e. a media-ring seal) is formed bythe contact of the sealing ring of the dish top with the media surfacecontained in the dish bottom when the media surface comes to rest on thesealing ring. This creates a headspace defined by the media surface, theinside wall of the sealing ring, and the inside top of the dish lid.

Furthermore, when the culture dish is utilized with the oxygen reducingagent such as a biocatalytic oxygen reducing agent, the oxygen reducingagent in the media reacts with the oxygen trapped in that headspace.This reaction renders the headspace sufficiently low in oxygen such thatmicroorganisms affected by the presence of oxygen can grow on the mediasurface typically within 24 to 48 hours when the dish is incubated at35° C. to 37° C. in an aerobic incubator. Any oxygen that intrudes intothe dish around the media ring-seal or through the plastic is removed bythe action of the reducing agent. The catalytic reducing agentfacilitates the design and function of this dish.

The media suitable for use in the present invention includes any solidtype media which can be inverted to form a media ring-seal. Solid mediagenerally consists of liquid media which have been solidified ("gelled")with an agent such as agar or gelatin. Examples of other known suitablegelling agents include alginate, gellan gum ("Gelrite™") and silica gel("Pluronic Polyol F127™"). The solid type media is of such a compositionto support growth of anaerobes, microaerophiles and facultative aerobes.

Further, the culture dish, i.e., "OxyDish™", of the present invention,is designed in certain embodiments so that it can be stacked in a stableconfiguration. The dish top has a stacking ring that interlocks with theadjacent dish top below it. The dish bottom, when the assembled dish isinverted and placed in a sealed position, rests (i.e., nests) betweenthe two adjacent dish tops. The functionality of the dish to establishand maintain an anaerobic environment is preserved and protected in thestack. The stackability of the culture dish increases the efficient useof incubator space. Stackability is also important for the mechanizedfilling of these dishes and shipment of dishes or of finished pre-made,plates to the microbiologist or end user.

The culture dish of the present invention, simplifies handling anaerobesby the microbiologist or laboratory technician. The culture dish, i.e.,"OxyDish™", can be opened and closed several times while continuing togenerate an anaerobic environment in the closed position. The speciallydesigned culture dish significantly increases the microbiologist'sefficiency by reducing and simplifying the number of manipulationsrequired to work with anaerobes. Furthermore, the microbiologist can nowtreat each culture dish and its microbial contents individually. Thisallows the microbiologist to make decisions based on his observations ofeach isolate or treatment, rather than having to wait for the slowestgrowing isolate in a group of culture dishes present in a sealable jar,bag, etc. In addition, the self-contained, environmentally controlledculture dish provides a secure environment for the microbe inside.

The foregoing has outlined some of the most pertinent objects of theinvention. These objects should be construed to be merely illustrativeof some of the more prominent features and applications of the intendedinvention. Many other beneficial results can be attained by applying thedisclosed invention in a different manner or by modifying the inventionwithin the scope of the disclosure. Accordingly, other objects and amore detailed understanding of the invention may be had by referring tothe drawings, the detailed description of the invention and the claimswhich follow below.

BRIEF DESCRIPTION OF THE DRAWINGS

The following is a brief description of the drawings which are presentedfor the purposes of illustrating the invention and not for purposes oflimiting the same.

FIG. 1 is a cross-sectional view of a two-part culture dish for growinganaerobic microorganisms shown in separated or exploded relation.

FIG. 2 is a cross-sectional view of the assembled culture dish of FIG. 1shown in what is generally referred to as a first or upright position.

FIG. 3 is a cross-sectional view of the assembled culture dish shown inwhat is generally referred to as a second or inverted position.

FIGS. 4A is a top view of a first component or bottom dish of theculture dish.

FIG. 4B is a side elevation view of the bottom dish.

FIG. 4C is a bottom view of the bottom dish.

FIG. 4D is an enlarged view of the side wall of the bottom dish.

FIGS. 5A is a top view of the second component or dish top or cover ofthe culture dish.

FIG. 5B is a side elevational view of the dish top.

FIG. 5C is a bottom view of the dish top.

FIG. 5D is a sectional view of the dish top taken generally along thelines 5D--5D of FIG. 5C.

FIG. 6 is a side elevational view of two assembled culture dishesstacked vertically in an upright position.

FIG. 7 is a side elevational view of three assembled culture dishesstacked vertically in an inverted position.

FIGS. 8A and 8B are photographs exhibiting growth of anaerobic organismsin the culture dish of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The culture dish of the present invention is designed to meet the strictrequirements of anaerobiosis while simplifying handling by themicrobiologist. As shown in the drawings, a culture dish 10 includes twoseparately configured parts. A first component or dish bottom 12receives a culture media 14 and a dish cover or top 16 defines a secondcomponent of the culture dish. The dish bottom and dish top selectivelycooperate to define a culture dish for growing microorganisms. Together,the dish bottom 12 and dish cover 16 define in a first position ororientation a covered Petri culture dish as shown in FIG. 2. This firstposition will be referred to as an upright position. When inverted (FIG.3), the dish bottom and dish cover alter their cooperative configurationto define a second position or orientation that forms an enclosedchamber or head space 80 in which anaerobic microorganisms 20 can becultivated.

The structural and functional details of the dish bottom 12 will bedescribed with reference to FIGS. 1-3, and more particularly withreference to FIGS. 4A-4D. The dish bottom is comprised of a generallyplanar base 22 and a circumferentially continuous side wall 24 extendinggenerally orthogonally from the base. For purposes of discussion only,the side wall will be described as extending upwardly from the base asillustrated in FIGS. 1, 2, and 4. It will be recognized, however, thatany directional description is merely for purposes of simplifying anunderstanding of the present invention.

In addition, the dish bottom 12 has a first or inner surface 26 thatfaces inwardly over the base and side wall toward the cavity defined bythe cup-shaped arrangement of the base and side wall. Likewise, a secondor outer surface 28 faces away from the cavity and encompasses theexterior surfaces of the base and side wall. The inner surface of theside wall is preferably divided by a lip 30 into first and secondportions 32, 34. The lip 30 can be optionally provided on the innersurface 26 of the side wall of the dish bottom and acts as a guide forindicating the fill height of the culture media 14. The first portion ofthe side wall defines an upper rim 36 and the second portion 34 joinsthe upper rim to the base 22. As illustrated, the base and side wall areshown as a one-piece construction such as a molded arrangement, althoughother equivalent configurations can be used without departing from thescope and intent of the invention.

The dish bottom 12 can be of any convenient dimension, and is usuallycircular so that this dimension is referenced as a diameter. Typically,the diameter of the dish bottom is about eight (8.0) to fifteen (15.0)cm. The depth of the dish bottom 12 defined by the height of the sidewall as it extends upwardly from the base can vary and is generallyabout 0.8 to 1.8 cm. In certain embodiments (FIG. 4C), the base 22 ofthe dish bottom 12 can be divided into two, three, four or more sections38 by sectional dividers, grid markings or other indicia 40 to enhancedifferential diagnostics of microorganisms (FIG. 4C).

The dish cover or top 16 (FIGS. 1 and 5A-5D) is sized to fit or conformover the dish bottom 12. The dish cover includes a top wall 50, firstand second side walls 52, 54, and a seal ring 56. The top wall isdisposed at approximately mid-height of the outer side wall 52 forreasons which will be described in greater detail below. In a mannersimilar to the dish bottom, the side walls 52, 54 are disposed generallyorthogonal to the top wall and are themselves radially spaced apart by agap or recess 58. The side walls are joined along one end by aninterconnecting wall 60 to define an inverted, generally J-shapedconfiguration when the culture dish components are disposed in the firstor upright position (FIGS. 1 and 2).

The dish cover has a first or inner surface 70 that generally faces thedish bottom when the individual components of the culture dish areassembled. A second or outer surface 72 extends over the exterior of thedish cover.

The seal ring 56 projects outwardly or downwardly from the inner surface70 of the top wall 50. The ring is circumferentially continuous andlocated along the radial periphery of the top wall. It alsointerconnects along its outer radial edge with the inner or second sidewall 54. The seal ring has a planar seal face 78 that cooperates withthe culture medium 14 in a dish bottom to define an anaerobicenvironment for growing microorganisms when disposed in an invertedposition (FIG. 3) and as will be described in greater detail below.

The recess 58 in the dish cover is defined between the first and secondside walls. When the dish cover 16 is assembled with the dish bottom 12and placed in an upright position (FIG. 2), the side wall 24 of the dishbottom is received in the recess 58 of the dish cover. The recess canvary in width depending upon the overall size and configuration of thedish cover and dish bottom.

Further, when the cover 16 is joined with the dish bottom 12 andpositioned in an upright position, the height of the side wall 52 of thedish cover is sufficient to keep the planar seal face 78 of the sealring 56 from contacting the medium surface 14. This allows the freshlypoured plate with molten agar, to cool and solidify before the mediasurface of the dish bottom 12 can rest on the seal ring in the dishcover 16 when the assembled culture dish 10 is inverted (FIG. 3). Thisfeature also provides a means for producing finished plates in acontinuous manner by mechanized means on a conveyor belt for large scalecommercial production while maintaining aseptic conditions.

When dish bottom 12 is filled with solidified media 14 and the assembledculture dish 10 is inverted, the solidified media surface will come intocontact with the seal ring 56 of the dish cover 16 forming a media-ringseal along the planar seal face 78 (see FIGS. 3 and 7). In this invertedconfiguration of the culture dish, an oxygen reducing agent present inthe media 14 will remove all of the oxygen that is trapped in the headspace of the enclosed chamber 80 formed between the surface of thesolidified media and the inner surface 70 of dish cover. The assembledculture dish can be incubated aerobically in the inverted position whileproducing an internal anaerobic environment for the growth of anaerobicmicroorganisms 20.

In addition, in certain embodiments of the invention, a raised area,dome or cavity 82 is present in the dish cover 16 (FIG. 1).Specifically, projecting outward from the top wall 50 of the dish coveris a dome 82 designed to contain an anaerobic gas (CO₂, etc.) generatingpad or element or an indicator strip (not shown). The dome 82 iscomprised essentially of a dome side wall 84 and a dome top wall 86,although a circular dome is the more preferred embodiment. It isunderstood by those skilled in the art that domes or cavities ofalternative shapes and sizes can be utilized with equal success.

In accordance with the illustrated embodiment, strengthening ribs 90 areperipherally spaced along side wall 52. The ribs are preferably equallyspaced about the circumference of the dish cover and protrude radiallyoutward from the exterior surface of the side wall. The ribs provideadditional rigidity and strength to the dish cover which is particularlyhelpful when the culture dishes are stacked in either the upright orinverted positions as shown in FIGS. 6 and 7.

The dish cover 16 can be of any convenient diameter so long as it mateswith the dimensions of the dish bottom 12. Typically, the dish cover 16is approximately nine (9.0) cm to sixteen (16.0) cm in diameter. Theseal ring 56 can be of any desired diameter and is generally about seven(7.0) cm to fourteen (14.0) cm in diameter and is centrally positionedrelative to the side wall 52 of the dish cover. The overall radialdimension of the seal ring 56 can vary with a preferred radial dimensionbeing about two-tenths (0.2) cm to one-half (0.5) cm.

Moreover, in some embodiments, the base 22 of the dish bottom 12contains an indicator ring 100 (FIG. 4A). The area or annulus 102between the indicator ring 100 of base and the side wall 24 identifiesthe area on the media surface 14 that the seal ring 56 will occupy whenthe assembled culture dish 10 is inverted. This area is not to be usedfor culturing microorganisms. Microbes present in this area will swarmaround the seal ring 56 when the dish top is placed in contact with theculture media.

The difference in the height of the side wall 52 of the dish cover inrelation to the height of the side wall 24 of the dish bottom can alsovary, with a height differential of about one-half (0.5) cm beingpreferred. The fill height 110 in FIG. 1 and FIG. 4D is the distancefrom the base 22 of the dish bottom 12 to the surface level of theculture media 14 and is variable. Typically, this height can betwo-tenths (0.2) cm to four-tenths (0.4) cm. The dimension from the topof the culture media 14 surface, which is generally indicated by theinner lip 30 to the top edge of the dish bottom is (D) and is determinedby the relationship D=A-C. (see FIG. 4D, wherein (A) is the total heightof the side wall 24 of the dish bottom 12 and (C) is the fill height ofthe culture media).

The seal ring 56 inside the dish cover extends a distance downward fromthe dish cover 16 equal to (E) in FIG. 5B which is determined by therelationship E=B-(Cmax+0.1 cm), where (B) is the total height of theside wall 52 of the dish cover and (Cmax) is the maximum fill height ofthe culture media 30. This assures that the seal ring 56 clears theculture medium surface by approximately one-tenth (0.1) cm when the dishbottom 12 is filled to its maximum level and the assembled culture dish10 is in its upright position (FIG. 2).

The distance between the top edge of the side wall 24 of the dish bottomand the upper extreme of the inner surface 70 of the dish cover 16 whenthe culture dish is oriented in the upright position is (F) in FIG. 6.In a preferred embodiment, F is determined by F=B-A, where (B) is thetotal height of the side wall 52 of the dish cover 16 and (A) is thetotal height of the side wall 24 of the dish bottom 12.

The depth of the headspace or enclosed chamber 80 below the mediasurface 38 formed when the assembled culture dish is inverted to form amedia-ring seal is determined by the dimension (G) in FIG. 7. Thisdimension can vary depending on the size of the dish top 16, buttypically ranges between two-tenths (0.2) cm to one-half (0.5) cm. Thedimension (H) in FIG. 7 is the height of the top wall from an upper edgeof the dish cover. It is determined according to the followingrelationship H=E-G, where (E) is the height of the inner side wall 54 ofthe dish cover and (G) is the height of the headspace 80.

The dish cover preferably includes one or more cut-out areas 112 (FIG.5B) in a portion of the side wall 52. These cut-out areas 112 facilitatethe grasping and separation of the dish bottom 12 from the dish cover inan assembled culture dish 10. The cut-out areas may be variably orconstantly spaced from each other in the side wall 52 of the dish cover.As shown, one preferred arrangement has two cut-out areas 112 in theside wall 52 that are equidistant from each other. Likewise, theparticular configuration of the cut-out areas may vary without departingfrom the scope and intent of this invention.

Moreover, in the preferred embodiment of the invention, the assembledculture dishes 10 are designed to be stacked one on top of another. Adish bottom 12 of one assembled culture dish is nestled between stackeddish covers 16 (see FIG. 6) in the upright positions. In this regard,each dish cover has a stacking ring or protruding rib 120 around theupper edge of the dish cover (FIG. 1). While the diameter of thestacking ring 120 can vary, it is generally about one-half (0.5) mm toone (1.0) mm less than the overall peripheral diameter of the dish cover16. This provides an outer radial ledge 122 upon which the bottom edgeof the side wall 52 of an adjacent dish cover rests when placed eitherin an upright (FIG. 6) or inverted position (FIG. 7). The projection ofthe stacking ring 120 is preferably about one-half (0.5) mm to one andone-half (1.5) mm in height. The stacking ring 120 prevents an adjacentdish cover from sliding laterally and upsetting the stacked arrangement(see, for example, FIGS. 6 and 7).

Similarly, the stacking ring 120 on the dish cover 16 radially containsor nests an adjacent dish bottom 12 when stacking is desired in anupright position (see FIG. 6). The stacking ring 120 defines a radialinner ledge 124 to impede slide-out of the enjoining dish bottom 12. Thestacking ring is preferably one-half (0.5) mm to (1.5) mm in width.

The minimum fill height (Cmin) to which the dish bottom 12 can be filledwith culture media 14 and have the media surface rest on the seal ring56 when the dish is in an inverted position is determined by Cmin=A-E,wherein (A) is the total height of the side wall 24 of the dish bottom12 and (E) is the height of the inner side wall 54 of the dish cover. Ifthe fill height of the culture media 14 is below this level, then theupper rim of dish bottom 12 rests in contact with the inner surface 70of the dish cover 16 rather than the media surface 14 resting on theseal ring 56 of the dish cover when the assembled culture dish isinverted. In this situation, there is no seal formed between the sealring 56 and the medium surface 14. The sealed headspace 80 is notformed. This condition renders the assembled culture dish 10 useless forone designed purpose of the culture dish which is to provide aself-contained environment for the isolation and growth ofmicroaerophiles and anaerobes.

A variant of the culture dish contains one or more perforations or pores132 in the dish bottom 12 for the purpose of controlling moisture insidethe headspace 80. The size of the pores 132 can vary but are usuallyabout one-tenth (0.1) cm to three-tenths (0.3) cm in diameter. Thenumber of pores 132 can vary from one (1) to eighty (80) or more andtheir location can be grouped or evenly spaced. The pores may be coveredwith an adhesive film (not shown) such as Mylar which retards thepassage of oxygen and can be sterilized in place when the dish issterilized. This film can be removed after the culture dish 10 is filledand before it is incubated. The pores provide a means to reduce thewater content of the media during incubation in a controlled manner.This reduces the condensate that forms inside the assembled culture dish10. Any oxygen infiltrating into the assembled culture dish 10 throughthese pores 132 must pass through the media 14 to get to the mediasurface where the microbes 20 are planted. The media 14 contains thebiocatalytic oxygen reducing agent and optionally one or more substratesthat removes the oxygen before it can reach the surface by this route.

The culture dish 10 is designed to be easily manufactured by knowninjection molding techniques. The dish top 16 and dish bottom 12 have nofeatures that prevent them from being ejected from a mold. The materialsof construction can vary but are preferably polystyrene, polycarbonate,or polystyrene-acrylonitrile. These are clear thermoplastics that areinexpensive, easy to mold, sterilizable by ethylene oxide or radiation,resilient to handling and resistant to chemical substances used inmicrobiological media. Styrene-acrylonitrile has the lowest oxygenpermeability of the three thermoplastics mentioned. All of the parts arepreferably transparent to permit observation of the anaerobic culturingprocess. However, pigments or dyes may be added to the polymericmaterials in order to produce different shades or colors. Further, as itis understood by those skilled in the art, ultra-violet light absorbersand other additives can be added to produce culture dishes having theproperties desired by the end user.

The assembled culture dish 10 can be opened by one of three methods:

A) The assembled and sealed culture dish 10 is placed upright on a benchtop. The dome 82 on the dish top 16 is depressed. The flexing of thedish top 16 causes the media-ring seal to part releasing and allowingthe dish bottom 12 to come to rest on the bench top.

B) The assembled and sealed culture dish 10 is gently struck onto thebench surface. This action breaks the media-ring seal which in turnreleases the dish bottom 12 and allows the dish bottom 12 to come torest on the bench top.

C) The assembled and sealed culture dish 10 is placed upright on a benchtop. The side walls 52 of the dish top 16, between cut-out areas, aregently flexed. This action causes the media-ring seal to part andreleases the dish bottom 12 to come to rest on the bench top.

The media-ring seal can be reformed simply by placing the dish top 16over dish bottom 12 and re-inverting the assembled culture dish 10.Gravity will cause the dish bottom 12 containing the solidified media tocome into contact with the seal ring. The substrate and/or oxygenreducing agent present in the media 14 will once again remove all of theoxygen trapped in the head space 80.

While the culture dish of the invention has been shown and describedherein as being particularly adapted for use in circular form, it is notdesired or intended to thus restrict the scope and utility of theimprovements by reason of such specific embodiments since the apparatusmay be of various shapes and sizes without departing from the invention.In addition, it is also contemplated that certain specific descriptivetechnology used herein shall be given the broadest possibleinterpretation consistent with the disclosure.

The biocatalytic oxygen reducing agents suitable for use in theinvention include known biocatalytic oxygen reducing agents such asglucose oxidase and catalase and the oxygen scavenging bacterial cellmembrane fragments disclosed in U.S. Pat. No. 4,476,224 entitled"Material and Method for Promoting the Growth of Anaerobic Bacteria",issued Oct. 9, 1984 to Howard I. Adler, Oak Ridge, Tennessee, one of theco-inventors of the present invention. The '224 patent is incorporatedherein by reference.

The '224 patent is directed to a method of removing dissolved oxygenfrom a nutrient medium for anaerobic bacteria through the use of sterilemembrane fragments derived from bacteria having membranes which containan electron transport system which reduces oxygen to water in thepresence of a hydrogen donor in the nutrient medium. It is known that agreat number of bacteria have cytoplasmic membranes which contain theelectron transport system that effectively reduces oxygen to water if asuitable hydrogen donor is present in the medium. Some of the bacterialsources identified in the '224 patent include Escherichia coli,Salmonella typhimurium, Gluconobacter oxydans, and Pseudomonasaeruginosa. These bacterial membranes have been highly effective inremoving oxygen from media and other aqueous and semi-solidenvironments.

The same oxygen reducing effects produces by the cell membrane fragmentsfrom the bacterial sources indicated above, are also present in themembrane of mitochondrial organelles of a large number of highernon-bacterial organisms. More particularly, a great number of fungi,yeasts, and plants and animals have mitochondria that reduces oxygen towater, if a suitable hydrogen donor is present in the medium. Some ofthe sources of oxygen reducing membranes from these mitochondria are:beef heart muscle, potato tuber, spinach, Saccharomyces, Neurospora,Aspergillus, Euglena and Chlamydomonas. The process of producing theuseful mitochondrial membrane fragments involves the following steps:

1. Yeast, fungal cells, algae and protozoa, having mitochondrialmembranes containing an electron transfer system which reduces oxygen towater, are grown under suitable conditions of active aeration and atemperature which is conducive to the growth of the cells, usually about20° C. to 45° C. in a broth media. Alternately, mitochondria may beobtained from the cells of animal or plant origin.

2. The cells are collected by centrifugation or filtration, and arewashed with distilled water.

3. For the preparation of crude mitochondrial membrane fragments, aconcentrated suspension of the cells is treated to break up the cellwalls and mitochondria. This is accomplished by known means, forexample, by ultrasonic treatment or by passing the suspension severaltimes through a French pressure cell at 20,000 psi.

4. The cellular debris is removed by low speed centrifugation or bymicrofiltration (cross-flow filtration).

5. The supernatant or filtrate is subjected to high speed centrifugation(175,000Xg at 5° C.) or ultrafiltration.

6. For the preparation of material of higher purity, the cells of step 2are suspended in a buffer containing 1.0M sucrose and are treated bymeans which break up the cell walls or membranes but leave themitochondria intact. This is accomplished by known means, for example,by ultrasonic treatment, passage through a French pressure cell at lowpressure, enzymatic digestion or high speed blending with glass beads.

7. The cellular debris from step 6 is removed by differentialcentrifugation or filtration.

8. The supernatant or retentate from step 7 is passed through a FrenchPress at 20,000 psi to break the mitochondria into small pieces.

9. Mitochondria debris from step 7 is removed by centrifugation at12,000Xg for approximately 15 minutes or by microfiltration.

10. The supernatant or filtrate from step 9 is subjected to high speedcentrifugation (175,000Xg at 5° C.) or ultrafiltration.

11. The pellet or retentate from step 5 (crude mitochondrial fragments)or the pellet or retentate from step 10 (purified mitochondrial membranefragments) are resuspended in a buffer solution at a pH of about 6.0 toabout 8.0. A preferred buffer solution is 0.02M solution ofN-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid (HEPES).

12. The membrane fragments in the buffer solution are then passed underpressure through a filter having openings of about 0.2 microns.

13. The suspension is then stored at about -20° C. for later use or itmay be freeze dried.

Furthermore, while many solidified medium do not require the addition ofa hydrogen donor in order for the enzyme system present in the membranefragments to reduce the oxygen present in the product to water, whensynthetic medium or medium failing to contain a hydrogen donatingsubstance are utilized, the addition of a hydrogen donor (i.e., anorganic substrate) may be necessary in order for the membrane fragmentsto perform their oxygen removing functions. Suitable hydrogen donors arelactic acid, succinic acid, alpha-glycerol phosphate, formic acid, malicacid, and where available, their corresponding salts.

The present invention is further illustrated by the following examples.It is to be understood that the present invention is not limited to theexamples, and various changes and modifications may be made in theinvention without departing from the spirit and scope thereof.

EXAMPLES Example 1

Growth of anaerobic microorganisms using the culture dish, i.e.,"OxyDish™" of the present invention and a biocatalytic oxygen reducingagent.

Nutrient agar is supplemented with sodium formate (15 mM), sodiumsuccinate (30 mM), sodium lactate (45 mM) and cysteine (0.025 g/100 ml).A biocatalytic oxygen reducing agent, EC-Oxyrase® (Oxyrase, Inc.,Mansfield, Ohio) is added to cooled (45° C. to 50° C.) but moltensterile medium to give a final concentration of 5 units/ml. 20 ml of theabove mixture is soon introduced into the bottom part of a culture dish,i.e., "OxyDish™". The top part of the culture dish, is placed over thefilled bottom part to prevent contaminants from entering the dish. Theagar in the bottom part cools to ambient temperature and solidifies. Thecovered dish is left standing to permit excess moisture to escape. Atthis point the dish may be sealed by inverting it to bring the agarsurface in the dish bottom into contact with the ring inside the dishtop.

A suspension of anaerobic microorganisms is spread on the surface of theagar medium that contains the biocatalytic oxygen reducing agent and itssubstrates. The dish is sealed by inverting it. The dish is then placedinto an aerobic incubator at 35° C. to 37° C. for 24 to 48 hours.Several dishes are stacked to form a stable column of dishes.

Assembled dishes can be handled and viewed at any time without breachingthe seal and losing the anaerobic environment inside the trappedheadspace. In this way, a particular culture dish, i.e., "OxyDish™" canbe selected at the earliest time when the microbial isolate has grownsufficiently for selection.

Using this technique with the culture dish, i.e., "OxyDish™" and abiocatalytic oxygen reducing agent, the following microorganisms havebeen grown: Clostridium tertium, C. difficile, C. perfringens, C.cadaveris, C. acetobutylicum, Bacteroides thetaiotaomicron, B. fragilis,B. distasonis, Escherichia coli, Fusobacterium varium, F. mortiferum, F.necrophorum, Peptostreptococcus magnus, P. anaerobius, P. nigra, P.intermedius, Lactobacillus casei, L. acidophilus, Eubacterium lentum,Bifidobacterium breve, and Streptococcus fecalis.

Example 2

Measurement of oxygen depletion in the headspace of the culture disheffect of the present invention by the biocatalytic oxygen reducingagent.

A hole is drilled in the base of the culture dish, i.e., "OxyDish™" anda gas tight septum is inserted. The base is then filled with 20 ml ofagar containing a biocatalytic oxygen reducing agent. The bottom issealed to the top by inverting the assembled dish and incubating it at37° C. Periodically, 50 ul samples of the gas in the headspace of thedish are sampled by inserting the tip of a 100 ul gas tight Hamiltonsyringe through the septum in the base of the dish. These samples areintroduced into an Oxygen Sensor (IT Corporation) and the concentrationof oxygen remaining in the headspace is determined. Using this method ithas been determined that all measurable oxygen, less than 10 pp billion,is removed from the head space in two to eight hours depending on theconcentration and configuration of the biocatalytic agent used. It hasalso been determined that the dish can be opened, resealed and, after asuitable incubation period, the head space again becomes anaerobic.

Example 3

Multiple opening and closing of the culture dish of the presentinvention and reestablishment of anaerobic environment.

A culture dish, i.e. "OxyDish™" of the present invention containing anutrient agar and the biocatalytic agent is streaked with an anaerobicorganism (Bacteroides thetaiotaomicron or B. fragilis) covering twoquadrants of the dish. After 24 hours of incubation at 37° C. the dishis opened, growth of the anaerobe is observed and a small quantity oforganism is streaked on the third quadrant of the dish. The dish isresealed and after 24 hours of incubation at 37° C., the dish isreopened and growth is observed in the third quadrant. A small amount ofgrowth from the third quadrant is streaked on the fourth quadrant. Thedish is resealed and after a 24 hour incubation at 37° C. it is reopenedand growth is observed in the fourth quadrant of the dish (see FIG. 8A).

Example 4

Rapid anaerobiosis of the agar layer containing a biocatalytic oxygenreducing agent as indicated by methylene blue.

An agar medium was made that contained water, 50 mM sodium lactate, and2.5 mg/ml of methylene blue. In the oxidized state methylene blue isblue in color. In the reduced state it is colorless. The agar was meltedand cooled to 45° C. It is blue in color. EC-Oxyrase® is added at 5units/ml and 20 ml is delivered to the bottom part of a culture dish,i.e., "OxyDish™". As soon as the agar has solidified, about 5 minuteslater, the culture dish, i.e., "OxyDish™" is sealed by inverting it. Atthis time the agar layer is blue in color. The culture dish, i.e.,"OxyDish™" is incubated at 37° C. and observed periodically. Soon aftersealing the culture dish, i.e., "OxyDish™", the agar layer begins tolighten in color. Within 30 minutes to 45 minutes of being put into theincubator, the medium is nearly white in appearance, but with a lightblue tinge of color. By 60 minutes of incubation the agar layer iswhite, which indicates that the agar layer is anaerobic shortly afterthe addition of EC-Oxyrase® to the medium.

Example 5

Use of methylene blue strip to indicate anaerobiosis in the culturedish.

A small rectangular piece of filter paper impregnated with methyleneblue at an alkaline pH is fixed to the inside of the dome in the top ofthe culture dish, i.e., "OxyDish™". The dish bottom contains nutrientagar and a biocatalytic oxygen reducing agent. The dish is sealed byinverting it thereby causing the agar surface to rest on the ring. Afterincubation at 37° C. for 8 hours or more, the blue color disappears fromthe filter paper. This indicates that the headspace of the culture dish,i.e., "OxyDish™" has become anaerobic.

Example 6

Use of glucose oxidase and catalase as the biocatalytic oxygen reducingagent.

Sterile Nutrient agar (Difco), supplemented with 1% glucose, is cooledto 45° C. and 1 unit of sterile filtered glucose oxidase/ml, and 1 unitof sterile filtered catalase (Sigma Biochemicals, 1994 Catalog, p 221and p 478) is added. 20 ml of this medium is deliver into a culturedish, i.e., "OxyDish™". After the agar has solidified, a small quantityof Bacteroides fragilis is streaked on the surface of the agar and thedish is sealed by inverting it. After 48 hours of incubation at 37° C.,growth of the anaerobic microorganisms is observed on the surface of theagar medium.

Example 7

Use of a filter pad with carbonate to generate CO₂ in the headspace.

A piece of filter paper saturated with a 1% sodium bicarbonate solutionand then dried is fixed to the inside of the dome in the culture dish,i.e., "OxyDish™" top. This filter paper is then covered by a 0.2 umembrane filter. The dish bottom is filled with 20 ml of Nutrient Agar(Difco) and a biocatalytic oxygen reducing agent, EC-Oxyrase® at 5units/ml and substrates. The agar surface is inoculated by streakingwith a small amount of Clostridium acetobutylicum, a microorganism thatrequires CO₂ for rapid colony development. Immediately before sealingthe dish, one drop of 0.1N HCl is placed on the membrane filter. Thedish is sealed by inverting it and placed into a 37° C. aerobicincubator. After 24 hours of incubation, growth of C. acetobutylicum canbe observed indicating that CO₂ was released from the sodium bicarbonateimpregnated filter paper into the headspace of the culture dish, i.e.,"OxyDish™".

Example 8

Relief of moisture through pores in the dish bottom.

Seventy-six holes of different sizes (large: 0.101 inch, medium: 0.086inch, and small: 0.059 inch) are drilled into a culture dish bottom. Thedish is filled with 40 ml of 1.5% agar. A standard dish cover or aBrewer Lid is fitted onto each dish bottom. The complete dish isweighed. The covered dish is incubated at 37° C. and the weighed attimed intervals. The loss of weight is taken as due to the loss ofmoisture, since the solidified agar is 98.5% water by weight. Therelative weight loss, net of control weight loss, is as follows:

    ______________________________________                                        Pore Size       24 hrs. 48 hrs.                                               ______________________________________                                        Small           6%      11%                                                   Medium          9%      14%                                                   Large           13%     24%                                                   ______________________________________                                    

This shows that the drying of the agar layer can be controlled duringincubation by the number and size of pores put into the dish bottom. Forall size holes, the molten agar did not escape through these holes. TheBrewer Lid covered dishes exhibited no moisture build up within thetrapped headspace for those dishes with holes in them.

Example 9

Comparison of Present Invention With Standard Methods for GrowingAnaerobes

The inoculum is prepared by selecting colonies of particular microbesfrom Wilkens-Chalgren blood agar plates. A loopful of growth issuspended in Brucella broth to a density of about 1.5×10⁸ colony formingunits per mL. The suspended microorganisms are put into wells of areplicator block. Sterile replica or pins are dipped into the wells ofthe block. The replicator pins are stamped onto the surface of agarmedium (Wilkens-Chalgren blood agar). Each pin is calibrated to deliverabout 1×10⁵ colony forming units per spot. This procedure is repeated toinoculate a controlled pattern of spots onto agar plates containingincreasing amounts of antibiotic. Appropriate control plates, that donot contain antibiotics, are included. After a short time, the spotsdry, the culture dish, i.e. the OxyDish, is sealed and incubatedaerobically. Standard plates, not containing the oxygen reducing agent,i.e. oxyrase®, and substrates, are incubated in anaerobic jars orchambers. After 48 hours of incubation at 35° C. the plates are scoredfor growth. The presence of growth on a plate containing antibioticindicates that the particular microbe is resistant to the level ofantibiotic in that plate. In this way, one can determine the antibioticsusceptibility profile of a large number of microbial specimens.

With respect to the rate and intensity of growth of a number ofdifficult anaerobes using the culture dish of the present invention andthe brocatalytic oxygen reducing agent compared to the standard methods,it was found that anaerobic microbes grew faster and to a greaterdensity with the present invention compared to standard anaerobicmethods. As shown below, this observation was particularly noticeablefor difficult to grow anaerobes.

    ______________________________________                                        Microbe      Standard Method                                                                           OxyDish Method                                       ______________________________________                                        Clostridium dificil                                                                        1+          3+                                                   Clostridium  4+          4+                                                   perfringens                                                                   Clostridium  1+          3+                                                   cadaveris                                                                     Bacteroides  3+          4+                                                   rhetaiotaomicron                                                              Bacteroides  3+          4+                                                   distasonis                                                                    Fusobacterium                                                                              2+          4+                                                   varium                                                                        Fusobacterium                                                                              2+          4+                                                   mortiferum                                                                    Fusobacterium                                                                              1+          3+                                                   necrophorum                                                                   Peptostreptococcus                                                                         2+          3+                                                   magnus                                                                        Peptostreptococcus                                                                         1+          3+                                                   anaerobius                                                                    Peptostreptococcus                                                                         1+          3+                                                   negra                                                                         Bifidobacterium                                                                            1+          3+                                                   breve                                                                         Prevotella   2+          3+                                                   intermedia                                                                    ______________________________________                                    

The invention has been described with reference to the preferredembodiments. Obviously, modifications and alterations will occur toothers upon reading and understanding the preceding detaileddescription. It is intended that the invention be construed as includingall such alterations and modifications insofar as they come within thescope of the claims and the equivalents thereof.

What is claimed:
 1. An apparatus for enumeration and cultivation ofanaerobes, microaerophiles and facultatively anaerobic microorganismscomprising:a first dish component having a base and side wall extendingtherefrom to define a cavity; and, a second dish component having a walland side wall extending therefrom that together cooperate with the firstdish component to define (i) a non-sealed assembly in a firstorientation of the dish components and (ii) a closed headspace betweenthe dish components in a second inverted orientation, and wherein thesecond dish component includes a circular rib protruding outwardly fromthe wall in a direction generally opposite that of the side wall.
 2. Theapparatus as defined in claim 1, wherein the rib is spaced radiallyinward from a peripheral edge of the second dish component and isadapted to receive a side wall of an adjacent second dish component whenplaced in stacked relation.
 3. The apparatus as defined in claim 1,wherein the rib is dimensioned to receive at least a portion of a baseof an adjacent first dish component when disposed in stacked relationand in the first orientation of the dish components.
 4. A culture dishfor growing anaerobic microorganisms comprising a dish bottom forreceiving a solidifiable culture media having a surface; and a dishcover adapted to be placed over said dish bottom, said dish cover havinga seal ring spaced from the media surface of the dish bottom when placedin an upright position and wherein said seal ring comes into contactwith the media surface in the dish bottom when the culture dish isinverted to form a seal that traps gas in a headspace between the mediasurface and the dish cover and wherein an oxygen reducing agent isincorporated into the media which reacts with oxygen in the media and inthe headspace thereby removing the oxygen and creating an environmentsuitable for growing anaerobic, microaerophilic and facultativeanaerobic microorganisms.
 5. A culture dish of claim 4, wherein the dishcover has a height sufficient to prevent the seal ring from contactingthe media surface when the dish cover is placed over the dish bottomafter receipt of the culture media.
 6. A culture dish of claim 4,wherein said seal ring of the dish cover has a depth of approximately 2mm to 5 mm, whereby the seal ring engages the media surface and definesa sealed headspace for growth of the microorganisms when the culturedish is inverted.
 7. A culture dish of claim 4, wherein said dish coverhas a dome located within the seal ring and the dome is adapted tocontain a gas generating element.
 8. A culture dish of claim 4, whereinsaid dish cover has a dome located within the seal ring and the dome isadapted to contain an anaerobic environment indicator agent.
 9. Aculture dish of claim 4, wherein said dish cover has a rib that extendsin a direction opposite to the seal rind to permit stacking of assembledculture dishes in a stable configuration.
 10. A culture dish of claim 4,wherein said dish bottom comprises a base and a projecting side wall,the side wall having a height sufficient to rest under the dish coverwhile preventing contact of the media surface of the dish bottom withthe seal ring of the dish cover when the culture dish is placed in theupright position and to allow the media surface to come into contactwith the seal ring of the dish cover when the culture dish is placed inthe inverted position.
 11. A culture dish of claim 10, wherein the dishbottom has a fill line on the side wall thereof to indicate the maximumfill height in the dish bottom.
 12. A culture dish of claim 4, whereinthe dish bottom contains pores having a diameter of approximately 0.1 mmto 0.4 mm to provide for evaporation of moisture from the mediacontained therein so as to prevent moisture build-up inside theheadspace.
 13. A culture dish of claim 4, wherein the dish cover isformed from one of polystyrene, polystyrene-acrylonitrile, andpolycarbonate.
 14. A culture dish of claim 4, wherein the dish bottom isformed from one of polystyrene, polystyrene-acrylonitrile, andpolycarbonate.
 15. A culture dish of claim 13, wherein the dish bottomand dish cover are formed from a transparent plastic.
 16. A culture dishof claim 4, wherein the dish cover has a stacking rib protruding from anexternal surface of the dish cover and in a direction opposite to theseal ring that facilitates stacking of the assembled culture dishes,dish cover to dish cover, with the dish bottoms nested between adjacentdish covers.
 17. A culture dish of claim 4, wherein the oxygen reducingagent is a biocatalytic oxygen reducing agent.
 18. A culture dish ofclaim 17, wherein the biocatalytic oxygen reducing agent is a membranefraction obtained from bacterial and mitochondrial sources.
 19. Aculture dish of claim 18, wherein the biocatalytic oxygen reducing agentincludes a glucose oxidase and catalase.
 20. A culture dish of claim 4,wherein the media is an agar media that supports the growth ofanaerobes, microaerophiles, and facultative aerobes.
 21. A culture dishof claim 4, wherein said dish cover has a side wall with at least onecut-out area to facilitate grasping and separation of the dish bottomfrom the dish cover.
 22. A method of cultivating and enumeratinganaerobes, microaerophiles and facultatively anaerobic microorganismscomprising:providing a culture dish having first and second dishcomponents that cooperate to define a cavity for cultivatingmicroorganisms; placing a solidified culture medium in the first dishcomponent; covering the first dish component containing the solidifiedculture medium with the second dish component, whereby the dishcomponents are configured in a first assembled orientation relative toone another; and inverting the assembled first and second dishcomponents, thereby forming a sealed headspace between the medium andthe second dish component for cultivating microorganisms between thefirst and second dish components in a second assembled orientation.